Methionine is a sulfur-containing amino acid which is essential in the nutrition of animals, and is often used as a feed additive to animals including poultry, pigs, cows, fish, equine species and even companion animals like dogs and cats. Historically, the methionine used for animal nutrition has been the racemic mixture of D and L methionine. Methionine is unusual in that most animals can utilize both the D and L forms of the amino acid. For all other essential amino acids, only the L form of the amino acid has nutritive value. Some specific animal studies have been completed which show benefits for feeding L-methionine as opposed to the racemic mixture. Faster absorption and better utilization in the muscle has been shown for some species under some selected feeding conditions. There are some specific applications outside of animal nutrition where the use of L-methionine is preferred. For example, L-methionine has known uses in human medicine and in the pharmaceutical industry. It is useful as a lipotropic agent and for the treatment of liver disease in animals. L-methionine and L-methionine derivatives are required for manufacturing therapeutic peptides, which are synthesized from single amino acids. Unfortunately, the production of the single isomer, L-methionine, is much more difficult and expensive as compared to producing the racemic mixture. Therefore, it would be very beneficial to establish a relatively inexpensive, industrial process for the production of L-methionine for human healthcare which also could be used for animal nutrition.
Several methods have been available for the production of L-methionine. For example, there is a process for the production of L-methionine by optically resolving DL-methionine prepared by a synthetic method (Pokorny et al., 1970, Phytochemistry 9:2175). Commercial production of L-methionine using acylase catalyzed cleavage of N-acetyl-D,L methionine is well known (see, for example, U.S. Pat. No. 4,827,029 and U.S. Pat. No. 6,656,710). These processes are fairly complex and therefore add significant additional cost in separating L-methionine from the racemic mixture. Processes based on selective crystallization are also known (see U.S. Pat. No. 6,673,942). It is also known to produce L-methionine by hydrolyzing proteins. Additionally, it is known to produce L-methionine by a microbiological process (e.g., fermentation).
There has been much published regarding the development of bacterial and yeast strains for L-methionine production. It is well known that L-methionine synthesis is tightly regulated in microorganisms. Consequently, the productivity of these microorganisms has been low with respect to methionine production. Kase and Nakayama isolated Coryneform mutants capable of producing 2 g methionine/liter (Agr Biol. Chem., 39(1), 153-160, 1975). Gomes and coworkers have also used classical mutagenesis techniques to isolate methionine analog resistant mutants of Corynebacterium lilium. Production of methionine by their isolate was shown to be much improved over the wild type starting strain, but much below commercial titres typically seen for other amino acids like lysine which is produced by fermentation. Commercial lysine fermentations typically reach titres approaching 100 g lysine/liter (see U.S. Pat. No. 5,268,293).
More recently, it has been reported by Moeckel, et al. in U.S. patent application No. 2002/0110878 that L-methionine production can be improved dramatically through the amplification of key genes in the methionine pathway. The expression of native Coryneform metA and metY genes was improved through the use of a specially constructed plasmid system. Shake flask fermentations of this strain reached a final methionine concentration of 16.0 grams of methionine/liter (g/L). Modification of other genes in the pathway in combination with the improvements in metA and metY should result in strains with even higher productivities. Using larger commercial fermentation systems which can support higher cell densities, these highly productive methionine-producing strains should be capable of producing methionine at high titre. Development of high methionine producing strains of E. coli is also being investigated. (See JP2000-139471 and 157267).
Because of the low solubility of methionine under normal fermentation conditions, its separation from whole cells and other fermentation broth component is a major issue which needs to be solved to be able to produce L-methionine economically. Typically, a neutral pH is preferred for the production of L-amino acids. For example, U.S. Pat. No. 3,729,381 teaches that a neutral pH is preferred to obtain high yield of L-methionine by fermentation (e.g., claim 3, and column 3, lines 28-31). U.S. Pat. No. 5,840,551 also teaches a method of producing L-amino acids by fermentation using neutral pH (e.g., see Example 1). The preferred fermentation temperature for organisms like Corynebacteria and E. coli is in the range of 30-37° C. Because of L-methionine's low solubility, both soluble and insoluble methionine fractions would exist in the broth. An effective separations process is needed to produce purified L-methionine from fermentation broth.